Pore-Forming Monopeptides as Exceptionally Active Anion Channels

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Pore-forming monopeptides as exceptionally active anion channels Changliang Ren, Fei Zeng, Jie Shen, Feng Chen, Arundhati Roy, Shaoyuan Zhou, Haisheng Ren, and Huaqiang Zeng J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b04657 • Publication Date (Web): 21 Jun 2018 Downloaded from http://pubs.acs.org on June 21, 2018

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Journal of the American Chemical Society

Pore-forming monopeptides as exceptionally active anion channels Changliang Ren,† Fei Zeng,† Jie Shen,† Feng Chen,† Arundhati Roy,† Shaoyuan Zhou,‡ Haisheng Ren‡ and Huaqiang Zeng*,† † ‡

Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669 College of Chemical Engineering, Sichuan University, Chengdu, China 610065

ABSTRACT: We describe here a unique family of pore-forming anion-transporting peptides, possessing a single-amino-acid-derived peptidic backbone that is the shortest among natural and synthetic pore-forming peptides. These monopeptides with built-in H-bonding capacity self-assemble into an H-bonded 1D columnar structure, presenting three types of exteriorly arranged hydrophobic side chains that closely mimic the overall topology of an -helix. Dynamic interactions among these side chains and membrane lipids proceed in a way likely similar to how -helix bundle is formed. This subsequently enables oligomerization of these rod-like structures to form ring-shaped ensembles of varying sizes with a pore size of smaller than 1.0 nm in diameter but sufficiently large for transporting anions across the membrane. The intrinsic high modularity in the backbone further allows rapid tuning in side chains for combinatorial optimization of channel’s ion transport activity, culminating in the discovery of an exceptionally active anion-transporting monopeptide 6L10 with an EC50 of 0.10 M for nitrate anions.

INTRODUCTION Naturally occurring pore-forming peptides (PFPs) belong to a large family of host defense peptides.1,2 They generally share a length of 12 – 50 amino acids in order to span the hydrophobic membrane region or form a cyclic structure enclosing a sufficiently large cavity, and carry at least one charge in its side chain or termini. With the addition of a long hydrophobic lipid moiety having 12 – 16 carbon atoms,3 PFPs can be shortened to as few as 2 - 4 amino acids.4,5 Nevertheless, natural1-4 or synthetic5-9 PFPs, which are made up of only a single amino acid residue and carry no sophisticated functional group, still remain elusive. We herein report such a unique yet extremely simple class of artificial pore-forming monopeptides to fill this missing gap (Figure 1a). Our molecular design is inspired by the way -helices interact with each other to form an -helix bundle (Figure 1b), a common motif that involves  3% of all amino acids in the known genomes for mediating intramolecular protein folding or intermolecular protein-protein interactions.11 More specifically, two or more helices could self-assemble into an -helix bundle via an interplay of non-covalent forces (van der Waals interactions, - stacking, H-bonds and salt bridges) among the side chains exteriorly arranged around the helical backbone (Figure 1b).10,12 These helix bundles, mostly consisting of 2 - 8 helices, play a particularly important role in associating multiple subunits to form oligomeric protein complexes of diverse types, including pore-forming toxins13 and protein channels14-19 in the cell membrane. They have also proven an excellent target for de novo protein design since 1980s,20-29 or key components in a range of supramolecular materials.30-32

Recently, we showed that amidating both the N- and C-termini of a single amino acid residue using simple groups (Fmoc, Cbz and alkyl chains, Figure 1a and 1c) gives rise to a rich family of phase-selective organogelators, which are able to rapidly and phase-selectively congeal crude oils of widely ranging viscosities in the presence of water at room temperature.33-35 Crystal structure of Fmoc-Phe-C4 reveals well-defined one-dimensional packing of the molecules via intermolecular H-bonds (Figure 1c).34 The high directionality of these H-bonds packs the same type of side chains (Fmoc, Phe and C4H9) to the same side. A closer examination shows that this H-bonded 1D scaffold with three types of exteriorly arrayed side chains topologically resembles that of an -helix (Figure 1b vs 1c and 1d), except that the 1D scaffold is non-covalently assembled via H-bonds and van der Waals forces while helically folded backbone in -helix is covalent in nature. Hinging on this great similarity in topology and the ability of -helices to form an -helix bundle, we hypothesized that monopeptides such as 5L10 might be able to self-assemble into an oligomeric ensemble, likely consisting of 3 8 H-bonded 1D structures, via van der Waals forces among their one-dimensionally aligned side chains (Figure 1d). The intrinsic high modularity involving R1-R3 groups should enable rapid screening and optimization of the ensemble’s properties in the membrane in a combinatorial manner. In a fortunate scenario, the combinatorially identified ensemble(s), alone or through dynamic interplay with membrane lipids, might achieve pore formation that alters the membrane permeability. In this report, we demonstrate that this hypothesis-driven strategy is highly effective, successfully delivering many monopeptides as excellent poreformers with high activity. We further demonstrate that these

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a

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b

Dimerization

GCN4-p1 c

Hydrophobic, H-bonding and Salt-Bridging residues

d Oligomerization

Fmoc (R1)

Phe (R2)

Fmoc

Fmoc-Phe-C4

C4 (R3)

Figure 1. -Helix-bundle-inspired molecular design of pore-forming monopeptides such as 5L10. a) Molecular construction of a pore-forming monopeptide library. b) Crystal structure of GCN4-P1, highlighting various types of non-covalent forces that drive the formation of a two-helix bundle.10 c) Crystal structure of Fmoc-Phe-C4, illustrating an H-bonded peptidic scaffold with directionally arrayed side chains (e.g., Fmoc, Phe and C4H9). d) Van der Waals interactions among the one-dimensionally aligned side chains might enable modularly tunable monopeptides (see a) to assemble into such as a hexameric ensemble, possessing a tubular cavity for ion transport. Note that ensembles of other sizes are also possible, and lipid molecules might also participate in the dynamic process of forming such ensembles.

pore-forming monopeptides conduct anions (chlorides, nitrate, etc), rather than cations, across the membrane.

RESULTS AND DISCUSSION Combinatorial identification of highly active pore-formers. Our first-round combinatorial screening of the ensembles’ iontransport profiles started with 100 monopeptide molecules (5 R1 x 4 R2 x 5 R3, Figure 1a) at a concentration of 5 M, using the pHsensitive HPTS (8-Hydroxypyrene-1,3,6-trisulfonic acid) assay (Figure 2a). In this assay, pH-sensitive HPTS dye (100 μM) and NaCl (100 mM) at pH 7 were encapsulated inside large unilamellar vesicles (LUVs), which were diluted into the same buffer containing no HPTS dye at pH 8 to generate a pH gradient of 7 to 8 across LUVs. Monitoring pH-dependent changes in fluorescence intensity of HPTS allows one to compare channel’s capacity in ion transport. From the results summarized in Figure 2b (For ion transport curves, see Figures S1-S5), two trends, which largely could be correlated to the lipophilicity of molecules, emerge. That is, molecules containing a very short alkyl chain (e.g., C4H9) at R2 position generally lack good ability to transport ions mostly with a fractional activity (RCl-) of < 5%, while ion transport activity apparently increases in the increasing order of Val ≈ Ala < Ile < Phe < Leu. In addition, 75, 49 and 26 out of 100

monopeptides display RCl- values of  5,  20 and  50%, respectively. These results suggest a majority of these hypothetic pore-forming peptide molecules indeed could assemble into a defined pore-forming structure of varying stabilities to facilitate ion transport across the membrane, thereby satisfactorily verifying the aforementioned hypothesis. Monopeptides that more strongly favour pore formation in membrane were then readily identified by lowering down the channel concentration from 5 to 2 M and further from 2 to 0.4 M. At 2 M, 7 molecules (e.g., 5L8, 5L10, 4L10, 3L10, 5F10, 4F10 and 3F10) could achieve an RCl- value of > 70% (Figures S6, S10 and S11). Among these molecules, 5L10, having an RClvalue of 65% at 0.4 M, turns out to be the most active (Figure 2c). Based on 5L10, our secondary screening logically looks into the impact the chain length in both R1 and R2 groups might have on ion transport activity. With R2 kept constant as C10H21, ion transport studies on nL10 (n = 1-9 and 11) establish 5L10 and 6L10 as the best among the nL10 series (Figures 2d and S12). With R1 kept constant as C5H11 or C6H13, continued investigations on two series of channel molecules (5Lm and 6Lm, m = 10, 12, 14 and 16, Figures 2e and S13) suggest a straight alkyl chain having 10 carbon atoms as the optimum side chain at the R 2 position.


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b

pH = 8 100 mM NaCl -

Cl

OH

Conc = 5 M

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HPTS

d 100

Conc = 0.4 M

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65% 5L10 60 40

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5F10 4F10 3L10 Blank/3F10

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Conc = 0.4 M

[EC50]5L10 = 0.32 μM [EC50]6L10 = 0.27 μM [EC50]7L10 = 0.33 μM

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85 40

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3 1 1L10 2L10

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Time (s)

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HPTS Assay Normalized FL intensity (RCl-, %)

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Conc = 0.4 M 90

5Lm 6Lm

60

30

0

C10 C12 C14 C16

Effect of R2

Figure 2. Ion-transport activities of 112 monopeptides determined using the HPTS assay. a) Schematic illustration of the LUV-based HPTS assay. HPTS refers to 8-Hydroxypyrene-1,3,6-trisulfonic acid whose fluorescence intensity increases with increasing pHs. b) Normalized ion transport activities (RCl-) for 100 monopeptides at 5 M over a duration of 5 min. RCl- = (I Cl- – I0)/(ITriton– I0) wherein ICl- and I0 are the ratiometric values of I460/I403 before addition of triton at t = 300 s, and ITriton is the ratiometric value of I460/I403 at t = 300 s right after addition of triton with internal/external buffers containing 100 mM NaCl and a pH gradient of 7 to 8 across LUVs. Comparative ion transport activities at 0.4 M among c) the seven most active monopeptides from the firstround screening of 100 library members, d) the nL10 series (n = 1-9 and 11) and e) the 5Lm and 6Lm series (m = 10, 12, 14 and 16).

Pore size and ion transport activity. Additional experiments using self-quenching CF dye (5(6)-carboxyfluorescein), rather than HPTS dye, trapped inside LUVs show that incorporation of the most active 5L10 and 6L10 at 4 M into LUVs lead to no leakage of CF dye inside LUVs (Figure S14). For comparison, melittin at 20 nM results in efflux of 71% CF dye from LUVs. From these results it can be inferred that (1) membrane integrity was maintained in the presence of 5L10 and 6L10 at high concentration and (2) the pore formed by 5L10 and 6L10 is smaller than the molecular size of CF dye (1.0 nm, Figure S14). Using Hill analyses, the EC50 values for reaching 50% transmembrane activity were determined to be 0.32 μM for 5L10 and 0.27 μM for 6L10, respectively (Figures 2d and S15a). Previously, the EC50 value of the most active giant unimolecular chloride channel, having a molecular weight of 3507 Da and carry eight linearly arrayed anion-binding units, was determined to be 0.88 μM under the identical conditions.36 On this basis, 6L10, having a molecular weight of 439 Da, actually is 2.3 times more active than the unimolecular chloride channel. Further, from the crystallographically determined intermolecular distance of 5.0 Å for Fmoc-Phe-C4 and 4.8 Å for 6L10 (Figure S16), 6-8 peptide molecules are required to span a typical hydrophobic membrane thickness of 28 Å for EYPC-based membrane.37 Taken together with the fact that only a portion of molecules will undergo self-

association to produce an H-bonded 1D columnar structure for facilitated ion transport, EC50 value in terms of effective channel concentration for 6L10 is estimated to be much lower than 0.045 μM (0.27 μM/6). At this concentration, the molar ratio of channel to lipid molecules is as low as 1:711 and comparable to the best natural or synthetic PFPs.7 This low molar ratio points to a high aptitude of 6L10 in forming a H-bonded 1D structure via Hbonds, which subsequently gives rise to a stable pore in the membrane via side chain interactions involving aliphatic side chains from 6L10 and perhaps also lipids. In addition, we have also looked into the effect of cholesterol at up to 30 mol% (relative to EYPC) on the ability of 6L10 to transport chloride anions. At [6L10] = 0.45 M where 6L10 elicits 94% ion transport activity in the absence of cholesterol, the ion transport activities decreases to 83, 80, 71, 68, 64 and 53% when cholesterol is increasingly present at 5, 10 15, 20, 25 and 30 mol%, respectively. OH−/Cl− as the major transport species through a channel mechanism. Aside from Cl-/OH- antiport mechanism illustrated in Figure 2a, other transport mechanisms and species, including Na+/H+ antiport, Na+/OH- symport and Cl-/H+ symport, might also be responsible for the observed increase in pH inside LUVs, leading to a time-dependent enhancement in fluorescence intensity of HPTS. To elucidate the most likely transport



pH = 8 100 mM MCl

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+

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pH = 7 100 mM NaCl HPTS

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[6L10] = 0.27 M

LiCl NaCl KCl RbCl CsCl

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100

200

300

Time (s)

Figure 3. Deciphering ion-transport mechanism and species. a) HPTS assay using varied extravesicular metal salts (MCl). b) SPQ assay using a chloridesensitive SPQ dye. c) HPTS assay using a proton carrier FCCP. d) HPTS assay using a potassium carrier valinomycin.

mechanism and species, a range of experiments was carried out on 6L10. First, extravesicular metal chloride salts were varied from LiCl to CsCl, and insignificant changes in fluorescence intensity of HPTS upon these variations is indicative of little involvement of metal ions in 6L10-mediated ion transport process (Figure 3a). Second, increasing additions of 6L10 from 0.06 to 0.4 M cause concentration-dependent increasing quenching of fluorescence of a chloride-sensitive SPQ dye ((6-methoxy-N-(3-sulfopropyl) quinolinium)), revealing chloride anion as one of the true transport species (Figure 3b). These two lines of evidences allow us to confidently conclude that, rather than the M+/H+ antiport or M+/OH- symport, either OH−/Cl− antiport (Figure 2a) or H+/Clsymport mechanism is the predominant mechanism operational in 6L10-mediated ion transport. A highly active proton carrier, FCCP (carbonyl cyanide 4(trifluoromethoxy)phenylhydrazone), was employed to differentiate the two possible mechanisms (Figure 3c). Relative to the transport efficiencies of 6% for FCCP alone and of 51% for 6L10 alone, a large enhancement of 22% in transport efficiency (e.g., 84% – 51% – (6% - 5%) by 6L10 in the presence of FCCP indicates a strong cooperative action between 6L10 and FCCP, and that Cl− is transported faster than H+.38 As for the relative transport rate between OH− and Cl−, we also performed the HPTS assay in the presence of valinomycin (VA), a K+-selective carrier (Figure 3d). We found that the ion transport activities of 6L10 (0.27 μM) in the presence and absence of of valinomycin (5 pM) differs very slightly by 4%, a difference that is identical to that between VA (9%) and blank (5%). These results not only suggest Cl− to be transported faster than OH− but also are consistent with a negligible role played by the H+/M+ antiport mechanism.38

a

2s

6L10 at 160 mV

100 fA

b

Current (fA) 120 -

γCl = 559.4 ± 6.8 fS

60

0 -200

-100

0 -60

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200

Voltage (mV)

-120

Figure 4. Single channel current measurement in planar lipid bilayers and determination of anion selectivity. a) Single channel current trace recorded in symmetric baths (cis chamber = trans chamber = 1 M KCl) at 160 mv for 6L10. b) Determination of Cl− conductance (γCl-) for 6L10 using a linear ohmic current-voltage (I-V).

Using Planar Lipid Bilayer Workstation (Warner Instruments), ion conducting behaviour of 6L10 was assessed in symmetric baths (cis chamber = trans chamber = 1 M KCl, Figure 4a). The evolved single channel current trace unequivocally confirms that 6L10-mediated chloride transport proceeds through a channel, rather than a carrier mechanism. Since the vertically aligned amide groups in the H-bonded 1D chain made up of eight 6L10 molecules apparently are not readily accessible for binding and mediating transport of a hydrated chloride anion (Figures 5a and


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Journal of the American Chemical Society b

a

14.3 Å MD simulation

R3 R1 R2 70 Å Computed structure of (6L10)8 in gas phase

H-bonds

MD-simulated structure of (6L10)8 in membrane

MD simulation

d

c

R3

R3

R1

R1

R2

R2 MD-simulated structure of 3×(6L10)8 in membrane

(6L10)8 from c

(6L10)8 from b

Figure 5. Computationally optimized structures for H-bonded (6L10)8 in gas phase and in lipid membrane. a) Computed structure of(6L10)8 using the COMPASS force field.45 b) MD-simulated structure of (6L10)8 inside a simulation box of 70 Å (w) x 70 Å (w) x 74 Å (h), comprising 128 POPC molecules and 4794 water molecules. c) MD-simulated structure of 3×(6L10)8 in DOPC membrane. d) Structural comparison between MD-simulated structures of (6L10)8 from b) and c). POPC = 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

S16a), we therefore proposed a barrel-stave pore model for chloride transport (Figure 1d). In this model, three to eight Hbonded 1D chains self-assemble into an oligomeric such as hexameric pore of < 1.0 nm in diameter, through interactions among alkyl chains at R1-R3 positions. Highly likely, these poreforming peptides and lipid molecules might also cooperate and remodel each other to achieve formation of a pore with optimum performance. In either scenario, it is anticipated that the pore formed this way is mostly lined up by hydrophobic alkyl chains. In view of opposite orientations of water molecules in the hydration shell of cations and anions, these alkyl chains, which line the pore and carry many slightly positively charged H-atoms, undoubtedly will interact more favourably, albeit weakly, with Oatoms from water molecules in the hydration shell of anions than with hydrated cations including protons. This reasoning is in good accord with our experimental finding on preferential transport of

Table 1. EC50 values for nL10 (n = 4 - 8), mL12 (m = 5, 6), 5A10, 3V10, 3I8 and 5F10 determined using the HPTS assay.

EC50 (uM) n valuea

a

4L10

5L10

6L10

7L10

8L10

5L12

0.53

0.32

0.27

0.33

0.39

0.36 3.8

4.7

3.3

3.5

3.4

3.1

6L12

5A10

3V10

3I8

5F10

EC50 (uM)

0.32

4.0

7.6

2.5

0.73

n valuea

3.5

3.8

1.3

1.5

3.5

Hill cofficient.

chloride over sodium ions or protons (Figure 3a) and with the fact that many other H-bonded monopeptide-based molecules show low or extremely low activity (Figure 1b), thereby suggesting an unlikelihood having Cl-/H+ co-transport as the major transport



species. Using single channel current traces recorded at various voltages, a linear ohmic current-voltage (I-V) curve could be generated from which the Cl− conductance (γCl-) for 6L10 was determined to be 559.4 ± 6.8 fS (Figures 4b and S17). The above set of experiments performed on 6L10 was also carried out on 5L10. Similar to 6L10, experimental findings on 5L10 are also supportive of (1) OH−/Cl− antiport as the main transport mechanism and species (Figure S18), (2) anion transport occurring through a channel mechanism with Cl− conductance of 552.8 ± 5.5 fS (Figure S19), and (3) high activity (EC50 = 0.32 μM, Figure S15a). We have further determined the EC50 values for the next best five pore-formers (e.g., 4L10, 7L10, 8L10, 5L12 and 6L12) as well as the best transporter from each of the other four amino acid series (e.g., 5A10 for alanine, 3V10 for valine, 3I8 for isoleucine and 5F10 for phenylalanine). As summarized in Table 1 and Figure S15, having EC50 values of 0.27 – 0.53 μM, the seven leucine derivatives are the most active among all 112 monopeptides tested in this work, and much better than the best four pore-formers respectively derived from the other four types of amino acids (A, V, I and F). The Hill coefficient values of 1.3 – 3.7 for these 11 channels (Table 2) are consistent with the fact that the functional channels are self-assembled from multiple molecules. Anion selectivity. The best two pore-formers 5L10 and 6L10 were chosen for further comparing their selectivity in anion transport toward five types of anions (e.g., Cl−, Br−, I−, NO3− and ClO4−). In these LUV-based experiments for determining anion selectivity, the same type and identical concentration of sodium salts (NaX at 100 mM) were used in both intra- and extravesicular regions with a proton gradient of pH 7 (inside) to pH 8 (outside). Comparison of EC50 values determined for five anions using Hill plots reveals the same selectivity trend, i.e. Cl− < I− < Br− < ClO4− < NO3−, for 5L10 and 6L10 with the nitrate anions as the most preferred transport species (Figures 6, S20 and S21). In more detail, the EC50 values are 0.32, 0.30, 0.24, 0.20 and 0.15 M for Cl−, I−, Br−, ClO4− and NO3− for 5L10, respectively. For 6L10, these values are 0.27, 0.20, 0.17, 0.15 and 0.10 M, respectively. As expected, 6L10 performs better than 5L10 in transporting all five types of anions. In the same way analyzed above for chloride transport, the effective channel concentration for 6L10 to transport NO3− anions is much lower than 0.017 μM (0.10 μM/6), which corresponds to an extremely low channel:lipid molar ratio of 1:1920. Conformation and stability of H-bonded structure in POPC membrane. The dynamic interactions between pore-forming peptides/proteins and lipids often play a decisive role in the poreforming process.8,39-43 For instance, majority of pore-forming peptides are unstructured in solution, and form functional pores only upon membrane binding/insertion.8,39,40 Even more interestingly, depending on lipid compositions, complete or partially formed pores are possible for pore-forming proteins such as membrane attack complex-perforin.41-43 In our current study, although the experimental evidences obtained using the HPTS assay and single channel current measurement unambiguously confirm the formation of well-defined channels for facilitated transport of anions across lipid membrane, it has remained quite challenging both experimentally and computationally to elucidate the pore structures or how the pores are formed. Nevertheless, it is

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Figure 6. EC50 values vs hydration energies for 5L10- and 6L10mediated transport of Cl−, Br−, I−, NO3− and ClO4− determined using the HPTS assay with both intra- and extravesicular regions containing the same type of NaX at 100 mM and a pH gradient of 7 to 8 across LUVs.

feasible to at least shed some lights into possible structural features of one-dimensionally aligned structure involving 6L10 in lipid membrane. In this regard, we first applied the COMPASS force field44 to optimize the H-bonded 1D structure, which consists of eight molecules of 6L10, and then embedded the optimized 1D structure in a bilayer of 128 POPC molecules (17152 atoms) solvated on two sides by 2 x 2397 water molecules, leading to a simulation system of 32062 atoms with a dimension of 70 Å (w) x 70 Å (w) x 74 Å (h) (Figure 5). Molecular Dynamics (MD) simulation using the CHARMM program,45-49 PME method50 and SHAKE algorithm51 was then carried out on this simulation system. After equilibration steps, the production run was carried out for 25 ns. The last 10 ns trajectories with 500 structural snapshots were used for analysing conformations of Hbonded structure in lipid membrane. Examining these 500 structures shows that (1) all side chains at R1, R2 and R3 positions, which are superimposable over each other in the initial structure (Figure 5a), become much less organized as evidenced from the highly populated structure shown in Figure 5b and 5d and (2) all eight molecules of 6L10 persistently remain H-bonded to each other in all 500 structures surveyed, demonstrating the reliability of intermolecular H-bonds in forming a H-bonded 1D structure able to span the hydrophobic membrane region. As shown in Figure S16a, 6L10 can have another energetically equivalent conformation where the angle formed by R1 and R2 groups is about 145. For this alternative structure, MD simulation converges on the same conclusions (Figure S16b). Further simulation on a trimeric ensemble comprising three Hbonded (6L10)8 in membrane supports the notion, as illustrated in Figure 1d, that intermolecular association among the projected side chains in H-bonded assembly (6L10)8 indeed could exist in lipid membrane (Figure 5c). Structural comparison compiled in Figure 5d indicates that a positive feedback mechanism might be operational during the self-assembly process. That is, inter-chain association helps to better organize side chains in a way to increase the self-association extent. However, given that (1) 6L10


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has two energetically equivalent conformations (Figures 5a and S16a) and (2) lipid molecules may also participate to modulate the assembled structures, we do anticipate the channel-forming process to be very complex, which certainly is beyond the scope of the present work.

CONCLUSION To summarize, in analogy to the way widely known -helix bundles are formed from -helices, we successfully designed and demonstrated a novel class of pore-forming monopeptides. These pore-formers are very unusual and unprecedented in that their backbone consists of only a single amino acid residue, amidated at two termini with simple alkyl chains to assist formation of Hbonded 1D columnar structure. These 1D structures resemble the topology of -helices and associate to generate pore-forming oligomeric ensembles conducible for highly efficient anion transport. The best of them exhibits exceptionally high anion transport activity when compared to other artificial anion channels elaborated over the past years.36,38,52-65 In particular, the EC50 value for 6L10-mediated transport of nitrate anions reaches as low as 0.10 M, or 0.017 M (0.05 mol% relative to lipid) in terms of effective channel concentration.

EXPERIMENTAL SECTION Proton transport study using the HPTS assay and EC50 measurements using the Hill analysis. Egg yolk L-αphosphatidylcholine (EYPC, 1 ml, 25 mg/mL in CHCl3, Avanti Polar Lipids, USA) and MeOH (1 mL) were mixed in a round-bottom flask. The mixed solvents were removed under reduced pressure at 40 oC. After drying the resulting film under high vacuum overnight at room temperature, the film was hydrated with 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid (HEPES) buffer solution (1 mL, 10 mM HEPES, 100 mM NaCl, pH = 7.0) containing a pH sensitive dye 8-hydrox-ypyrene1,3,6-trisulfonic acid (HPTS, 1 mM) at room temperature for 60 minutes to give a milky suspension. The mixture was then subjected to 12 freezethaw cycles: freezing in liquid N2 for 1 minute and heating 37 oC water bath for 1.5 minutes. The vesicle suspension was extruded through polycarbonate membrane (0.1 μm) to produce a homogeneous suspension of large unilamellar vesicles (LUVs) of about 120 nm in diameter with HPTS encapsulated inside. The unencapsulated HPTS dye was separated from the LUVs by using size exclusion chromatography (stationary phase: Sephadex G-50, GE Healthcare, USA, mobile phase: HEPES buffer with 100 Mm NaCl) and diluted with the mobile phase to yield 12.8 mL of 2.5 mM lipid stock solution. The HPTS-containing LUV suspension (25 μL, 2.5 mM in 10 mM HEPES buffer containing 100 mM NaCl at pH = 7.0) was added to a HEPES buffer solution (1.93 mL, 10 mM HEPES, 100 mM NaCl at pH = 8.0) to create a pH gradient for ion transport study. A solution of channel molecules in DMSO was then injected into the suspension under gentle stirring. Upon the addition of channels, the emission of HPTS was immediately monitored at 510 nm with excitations at both 460 and 403 nm recorded simultaneously for 300 seconds using fluorescence spectrophotometer (Hitachi, Model F-7100, Japan) after which time an aqueous solution of Triton X-100 (20 μL, 20% v/v) was immediately added to achieve the maximum change in dye fluorescence emission. The final transport trace was obtained, after subtracting background intensity at t = 0, as a ratiometric value of I460/I403 and normalized based on the ratiometric value of I460/I403 after addition of triton. The fractional changes RCl- was calculated for each curve using the normalized value of I460/I403 at 300 seconds before the addition of triton, with triton with ratiometric value of I460/I403 at t = 0 s as 0% and that of

I460/I403 at t = 300 s (obtained after addition of triton) as 100%. Fitting the fractional transmembrane activity RCl- vs channel concentration using the Hill equation: Y=1/(1+ (EC50/[C])n) gave the Hill coefficient n and EC50 values. The HPTS assay for cation selectivity. The HPTS-containing LUV suspension (25 μL, 2.5 mM in 10 mM HEPES buffer containing 100 mM NaCl at pH = 7.0) was added to a HEPES buffer solution (1.93 mL, 10 mM HEPES, 100 mM MCl at pH = 8.0, where M+= Li+, Na+, K+, Rb+, and Cs+) to create a pH gradient for ion transport study. A solution of monopeptide molecules 5L10 or 6L10 at a final concentration of 0.32 M or 0.27 M (EC50) in DMSO was then injected into the suspension under gentle stirring. Upon the addition of channels, the emission of HPTS was immediately monitored at 510 nm with excitations at both 460 and 403 nm recorded simultaneously for 300 seconds using fluorescence spectrophotometer (Hitachi, Model F-7100, Japan) after which time an aqueous solution of Triton X-100 (20 μL, 20% v/v) was immediately added to achieve the maximum change in dye fluorescence emission. The final transport trace was obtained as a ratiometric value of I460/I403 and normalized based on the ratiometric value of I460/I403 after addition of triton. The HPTS assay for anion selectivity. The HPTS-containing LUV suspension (25 μL, 2.5 mM in 10 mM HEPES buffer containing 100 mM NaX where X-= Cl-, Br-, I-, NO3- and ClO4- at pH = 7.0) was added to a HEPES buffer solution (1.93 mL, 10 mM HEPES, 100 mM NaX, where X-= Cl-, Br-, I-, NO3- and ClO4- at pH= 8.0) to create a pH gradient for ion transport study. A solution of 5L10 or 6L10 at a final concentration of 0.32 M or 0.27M (EC50) in DMSO was then injected into the suspension under gentle stirring. Upon the addition of poreforming monopeptide molecules, the emission of HPTS was immediately monitored at 510 nm with excitations at both 460 and 403 nm recorded simultaneously for 300 seconds using fluorescence spectrophotometer (Hitachi, Model F-7100, Japan) after which time an aqueous solution of Triton X-100 (20 μL, 20% v/v) was immediately added to achieve the maximum change in dye fluorescence emission. The final transport trace was obtained as a ratiometric value of I460/I403 and normalized based on the ratiometric value of I460/I403 after addition of triton.

The HPTS assay in the presence of FCCP (carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone). The HPTS-containing LUV suspension (25 μL, 2.5 mM in 10 mM HEPES buffer containing 100 mM NaCl at pH = 7.0) was added to a HEPES buffer solution (1.93 mL, 10 mM HEPES, 100 mM NaCl) to create a pH gradient for ion transport study. A solution of FCCP (0.5 M) and 5L10 (0.32 M) or 6L10 (0.27 M) in DMSO was then injected into the suspension under gentle stirring at 20 s and 70 s, respectively. Upon the addition of poreforming monopeptide molecules, the emission of HPTS was immediately monitored at 510 nm with excitations at both 460 and 403 nm recorded simultaneously for 300 seconds using fluorescence spectrophotometer (Hitachi, Model F-7100, Japan). 300 s later, aqueous solution of Triton X100 (20 μL, 20% v/v) was immediately added to achieve the maximum change in dye fluorescence emission. The final transport trace was obtained as a ratiometric value of I460/I403 and normalized based on the ratiometric value of I460/I403 after addition of triton. The HPTS assay in the presence of valinomycin (VA). The HPTS-containing LUV suspension (25 μL, 2.5 mM in 10 mM HEPES buffer containing 100 mM NaCl at pH = 7.0) was added to a HEPES buffer solution (1.93 mL, 10 mM HEPES, 100 mM NaCl) to create a pH gradient for ion transport study. A solution of valinomycin (5 pM) and 5L10 (0.32 M) or 6L10 (0.27 M) in DMSO was then injected into the suspension under gentle stirring at 20 s and 70 s, respectively. Upon the addition of pore-forming monopeptide molecules, the emission of HPTS was immediately monitored at 510 nm with excitations at both 460 and 403 nm recorded simultaneously for 300 seconds using fluorescence spectrophotometer (Hitachi, Model F-7100, Japan) after which time an aqueous solution of Triton X-100 (20 μL, 20% v/v) was immediately



added to achieve the maximum change in dye fluorescence emission. The final transport trace was obtained as a ratiometric value of I460/I403 and normalized based on the ratiometric value of I460/I403 after addition of triton.

Membrane leaking and pore size determination using CF dye. Egg yolk L-α-phosphatidylcholine (EYPC, 1 ml, 25 mg/mL in CHCl3, Avanti Polar Lipids, USA) and MeOH (1 mL) were mixed in a roundbottom flask. The mixed solvents were removed under reduced pressure at 40 oC. After drying the resulting film under high vacuum overnight at room temperature, the film was hydrated with HEPES buffer solution (1 mL, 10 mM HEPES, 100 mM NaCl, pH = 7.5) containing a 5(6)fluorescein (CF, 50 mM) at room temperature for 60 minutes to give a milky suspension. The mixture was then subjected to 12 freeze-thaw cycles: freezing in liquid N2 for 1 minute and heating at 37 oC in water bath for 1.5 minutes. The vesicle suspension was extruded through polycarbonate membrane (0.1 μm) to produce a homogeneous suspension of large unilamellar vesicles (LUVs) of about 120 nm in diameter with CF encapsulated inside. The free unencapsulated CF dye was separated from the LUVs by using size exclusion chromatography (stationary phase: Sephadex G-50, GE Healthcare, USA, mobile phase: HEPES buffer with 100 mM NaCl) and diluted with the mobile phase to yield 12.8 mL of 2.5 mM lipid stock solution. The CF-containing LUV suspension (25 μL, 2.5 mM in 10 mM HEPES buffer containing 100 mM NaCl at pH = 7.5) was added to a HEPES buffer solution (1.93 mL, 10 mM HEPES, 100 mM NaCl at pH = 7.5) to create a concentration gradient for CF dye transport study. A solution of 5L10 or 6L10 (4 M) or natural poreforming peptide Melittin in DMSO at different concentrations was then injected into the suspension under gentle stirring. Upon the addition of pore-forming monopeptide molecules, the emission of CF was immediately monitored at 517 nm with excitations at 492 nm for 300 seconds using fluorescence spectrophotometer (Hitachi, Model F-7100, Japan) after which time an aqueous solution of Triton X-100 (20 μL, 20% v/v) was immediately added to completely destruct the chloride gradient. The final transport trace was obtained by normalizing the fluorescence intensity using the equation of If = [(It- I0)/(I1- I0)] where, If = Fractional emission intensity, It = Fluorescence intensity at time t, I1 = Fluorescence intensity after addition of Triton X-100, and I0 = Initial fluorescence intensity . Chloride transport using the SPQ Assay. Egg yolk L-αphosphatidylcholine (EYPC, 1 ml, 25 mg/mL in CHCl3, Avanti Polar Lipids, USA) and MeOH (1 mL) were mixed in a round-bottom flask. The mixed solvents were removed under reduced pressure at 40 oC. After drying the resulting film under high vacuum overnight at room temperature, the film was hydrated NaNO3 solution (1 mL, 200 mM) containing a Cl--sensitive dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) (0.5 mM) in thermostatic shaker-incubator at room temperature for 60 minutes to give a milky suspension. The mixture was then subjected to 12 freeze-thaw cycles: freezing in liquid N2 for 1 minute and heating at 37 o C in water bath for 1.5 minutes. The vesicle suspension was extruded through polycarbonate membrane (0.1 μm) to produce a homogeneous suspension of large unilamellar vesicles (LUVs) of about 120 nm in diameter with SPQ encapsulated inside. The unencapsulated HPTS dye was separated from the LUVs by using size exclusion chromatography (stationary phase: Sephadex G-50, GE Healthcare, USA, mobile phase: 200 mM NaNO3) and diluted with the mobile phase to yield 12.8 mL of 2.5 mM lipid stock solution. The SPQ-containing LUV suspension (25 μL, 2.5 mM in 200 mM NaNO3) was added to a NaCl solution (1.93 mL, 200 mM) to create an extravesicular chloride gradient. A solution of monopeptide molecule 5L10 or 6L10 in DMSO at different concentrations was then injected into the suspension under gentle stirring. Upon the addition of pore-forming monopeptide molecules, the emission of SPQ was immediately monitored at 430 nm with excitations at 360 nm for 300 seconds using fluorescence spectrophotometer (Hitachi, Model F-7100, Japan) after which time an

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aqueous solution of Triton X-100 (20 μL, 20% v/v) was immediately added to completely destruct the chloride gradient. The final transport trace was obtained by normalizing the fluorescence intensity using the equation of If = [(It - I1)/(I0 - I1)] where, If = Fractional emission intensity, It = Fluorescence intensity at time t, I1 = Fluorescence intensity after addition of Triton X-100, and I0 = Initial fluorescence intensity .

Single channel current measurement in planar lipid bilayers. The chloroform solution of 1,2-diphytanoyl-sn-glycero-3-phosphocholine (diPhyPC, 10 mg/ml, 20 uL) was evaporated using nitrogen gas to form a thin film and re-dissolved in n-decane (8 uL). 0.2 L of this n-decane solution was injected into the aperture (diameter = 200 um) of the Delrin® cup (Warner Instruments, Hamden, CT) with the n-decane removed using nitrogen gas. In a typical experiment for conductance measurement, both the chamber (cis side) and Delrin cup (trans side) were filled with an aqueous KCl solution (1.0 M, 1.0 mL). Ag-AgCl electrodes were inserted into the two solutions with the cis chamber grounded. Planar lipid bilayer was formed by painting 0.3 L of the lipid-containing n-decane solution around the n-decane-pretreated aperture. Successful formation of planar lipid bilayers can be established with a capacitance value ranging from 80-120 pF. Samples (5L10 or 6L10) in THF (0.3-1.0 L) were added to the cis compartment to reach a final concentration of around 10 -8 M and the solution was stirred for a few min until a single current trace appeared. These single channel currents were then measured using a Warner BC535D bilayer clamp amplifier, collected by PatchMaster (HEKA) with a sample interval at 5 kHz and filtered with an 8-pole Bessel filter at 1 kHz (HEKA). The data were analysed by FitMaster (HEKA) with a digital filter at 100 Hz. Plotting current trace vs voltage yielded chloride conductance (γCl-). Molecular modeling using the COMPASS force field. The COMPASS force field (condensed-phase optimized molecular potentials for atomistic simulation studies) developed by Sun44 was used to optimize the geometry and to calculate the energy of all molecules. The COMPASS force field is based on state-of-the-art ab initio and empirical parameterization techniques with the valence parameters and atomic partial charges supported by ab initio data, and the van der Waals (vdW) parameters derived by fitting the experimental data of cohesive energies and equilibrium densities. The convergence tolerance is 2 × 10 −5 kcal/mol for the energy, 0.001 kcal/mol/Å for the force, 0.001 GPa for the stress, and 10−5 Å for the displacement. The Ewald method is used for calculating the electrostatic and the van der Waals terms. The accuracy is 10−5 kcal/mol. The repulsive cutoff is 6 Å for the van der Waals term. For the periodical structure, the box vector along the stacking direction is also optimized together with the molecules. Molecular dynamics simulations. Membrane builder in CHARMGUI45,46 is used to build the initial structure. The protocol comprises six steps as described by Jo et al,47 which are sequentially performed in the following order: objects reading, objects orientation, system size determination, building of lipid bilayer, assembly of lipid bilayer, and system equilibrium. In this work, the H-bonded structure, consisting of eight molecules of L8 (528 atoms), is placed in the center of the membrane made up of 128 molecules of 1-palmitoyl-2-oleoyl-sn-glycero3-phosphocholine (POPC) and a total of 17152 atoms. The membrane is then placed in a box of 70Å x 70 Å in width and 74 Å in height. 4794 water molecules are placed on the top side and bottom side of the membrane (2397 each side). Counter KCl ions were added to produce an ion concentration of 0.15 M. The simulation used the CHARMM36 (C36) force filed48 for lipids, CHARMM General Force Field (CGenFF)48 for the repeating unit of L8 and the CHARMM TIP3P water model. 49 The periodic boundary condition (PBC) were employed and the particle mesh Ewald (PME) method50 was used for long-range electrostatic interactions. The simulation time step was set to 2 fs in conjunction with the SHAKE algorithm51 to constrain the covalent bonds involving hydrogen atoms. The constructed system is first relaxed through molecular mechanics (MM) minimization of 20000 steps, then heated to 303.15 K using 50 ps


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NPT molecular dynamics (MD) simulations, and finally equilibrated using 200 ps NPT MD simulations. During MD simulations, the pressure was maintained at 1 bar. After equilibration steps, the production run of simulation was performed for 25 ns and the last 10 ns trajectories with 500 structures were used for analysing conformation and stability of Hboned structure.

ASSOCIATED CONTENT Supporting Information. Synthetic procedures for 112 monopeptides as well as a full set of characterization data including 1H NMR, 13C NMR, MS, ion transport study, CF assay, single channel current experiment and computational details. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author [email protected]

ORCID Huaqiang Zeng: 0000-0002-8246-2000 Arundhati Roy: 0000-0003-1129-1993

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore).

REFERENCES (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13)

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Graphic for the Table of Contents X-

M+

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Pore-Forming Monopeptides as Exceptionally Active Anion Channels

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